This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Yellow fever virus (YFV) vaccination has been found to cause severe viscerotropic disease in a substantial number of patients 60 years of age (incidence rate is approximately 1:50,000 doses administered) and these cases result in approximately 50% mortality. Increased monitoring efforts have also documented several cases of vaccine-related fatalities in young, otherwise healthy adults with no known pre-existing immune deficiencies. To date, there is no alternative to live YFV vaccination. In this proposal, we will use a proprietary new technology to develop an inactivated vaccine formulation that can be used to immunize vulnerable populations such as infants and elderly, in addition to other healthy populations. Our preliminary studies demonstrate that this vaccine approach is feasible and highly immunogenic. In this project, we will evaluate candidate vaccine formulations, perform scale-up development, and test in vivo efficacy against lethal YFV infection. To date, we have developed a novel approach to quantitating non-plaqueable strains of yellow fever virus using a flow cytometry-based limiting dilution assay. With this assay in hand, we have now grown and titrated new stocks of the virulent Dakar 1279 strain of yellow fever and initiated in vivo challenge studies in rhesus macaques. In addition, we have vaccinated groups of animals with an H2O2-inactivated YFV vaccine and in the coming weeks/months, we will challenge the animals with a lethal dose of Dakar 1279 and determine if our 2nd generation YFV vaccine is protective against lethal challenge.